The effects of various neurotransmitters on phosphoinositide hydrolysis, mobilization of Ca2+ and... more The effects of various neurotransmitters on phosphoinositide hydrolysis, mobilization of Ca2+ and release of [3H]-norepinephrine ([3H]-NE) were studied in cultures of sympathetic neurons of chick embryos. [3H]-inositol-1,4,5-triphosphate ([3H]-IP3) was increased in sympathetic neurons by acetylcholine (ACh), muscarine and serotonin (5-HT). Dopamine and norepinephrine did not stimulate phosphoinositide hydrolysis. Intracellular concentration of free Ca2+ ([Ca2+]i) was measured in Indo-1-loaded sympathetic neurons at rest and after addition of test agents. Measurements were made in the cell body and growth cone regions since Ca2+ mobilization is known to be different in different regions of the sympathetic neurons. ACh (nicotinic component was blocked by hexamethonium) and 5-HT failed to increase the [Ca2+]i, in the cell body as well as in the growth cone. The spontaneous release of [3H]-NE was not affected by ACh and 5-HT. Caffeine increased the [Ca2+]i only in the cell body but not ...
The Journal of pharmacology and experimental therapeutics, 2000
A novel series of N-substituted 4-ureido-5,7-dichloro-quinolines were synthesized to contain phar... more A novel series of N-substituted 4-ureido-5,7-dichloro-quinolines were synthesized to contain pharmacophores directed at voltage-sensitive sodium channels (VSNaCs) and N-methyl-D-aspartate (NMDA) receptors. These compounds were shown to act in a use-dependent manner as antagonists of VSNaCs and to act as selective competitive antagonists at the strychnine-insensitive glycine recognition site of NMDA receptors. These agents had little or no effect on alpha-adrenergic receptors, other glutamate receptors, or sites other than the glycine site on the NMDA receptor, and did not block voltage-sensitive calcium channels in vitro. In vivo, the compounds were active in preventing or reducing the signs and symptoms of neurohyperexcitability and had anxiolytic properties. Unlike benzodiazepines, N-substituted 4-ureido-5, 7-dichloro-quinolines showed little interaction with the sedative effects of ethanol, but were effective in controlling ethanol withdrawal seizures. The combined actions of the...
Alcohol research & health : the journal of the National Institute on Alcohol Abuse and Alcoholism, 2008
Researchers from a wide variety of backgrounds and with a broad range of goals have utilized high... more Researchers from a wide variety of backgrounds and with a broad range of goals have utilized high-throughput screening technologies (i.e., microarray technologies) to identify candidate genes that may be associated with an observable characteristic or behavior (i.e., phenotype) of interest. However, the initial microarray analyses typically also yield many genes that are not related to the phenotype of interest. Therefore, additional analyses are necessary to select the most likely candidates and eventually identify one or more genes that actually underlie that phenotype. After briefly explaining how microarray data are generated, this article describes one approach to narrowing down the resulting candidate genes and a database that can help in this analysis.
Alcohol research & health : the journal of the National Institute on Alcohol Abuse and Alcoholism, 2008
The development of alcohol dependence is posited to involve numerous changes in brain chemistry (... more The development of alcohol dependence is posited to involve numerous changes in brain chemistry (i.e., neurotransmission) that lead to physiological signs of withdrawal upon abstinence from alcohol as well as promote vulnerability to relapse in dependent people. These neuroadaptive changes often occur in those brain neurotransmission systems that are most sensitive to the acute, initial effects of alcohol and/or contribute to a person’s initial alcohol consumption. Studies of these neuroadaptive changes have been aided by the development of animal models of alcohol dependence, withdrawal, and relapse behavior. These animal models, as well as findings obtained in humans, have shed light on the effects that acute and chronic alcohol exposure have on signaling systems involving the neurotransmitters glutamate, γaminobutyric acid (GABA), dopamine, and serotonin, as well as on other signaling molecules, including endogenous opioids and corticotrophin-releasing factor (CRF). Adaptation to...
Alcohol and alcoholism (Oxford, Oxfordshire). Supplement, 1994
Ethanol inhibits the function of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor in... more Ethanol inhibits the function of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor in various neuronal systems, but the mechanism of the inhibition has not been elucidated. Previous work, using primary cultures of rat cerebellar granule cells, showed that both exposure to alcohol and activation of protein kinase C (PKC) by the phorbol ester PMA reduced the potency of the co-agonist, glycine, to enhance NMDA receptor function (measured as an increase in intracellular Ca2+), resulting in inhibition of the NMDA response at low glycine concentrations. Inhibition of NMDA receptor function by PMA and ethanol could also be overcome by PKC antagonists, implicating PKC in the inhibitory effect of ethanol. We have now compared the effects of ethanol and PKC activation of NMDA receptor function in primary cultures of rat cerebral cortical cells. The receptor in these cells was much less sensitive to ethanol inhibition, and the inhibition was not overcome by high concentrations of g...
Proceedings of the National Academy of Sciences, 1993
The concentration of cytosolic free Ca2+ ([Ca2+]i) and the release of tritiated norepinephrine ([... more The concentration of cytosolic free Ca2+ ([Ca2+]i) and the release of tritiated norepinephrine ([3H]NE) were monitored during Ba2+ stimulation of sympathetic neurons cultured from chick embryos. Ba2+ (2.5 mM in Ca(2+)-free medium) caused a rise in [Ca2+]i in all regions (cell bodies, neurites, and growth cones) of sympathetic neurons and evoked [3H]NE release in the absence of other stimuli. The increase in [Ca2+]i and release of [3H]NE were sustained for up to 30 min in the presence of Ba2+. When Ba(2+)-stimulated cells were immediately washed in Ca(2+)-free Ba(2+)-free EGTA solution, both the elevated [Ca2+]i and [3H]NE release returned to basal levels, with similar, fast, time courses. Ba2+ also blocked Ca2+ efflux from neurons loaded with 45Ca. We conclude from the parallel effects of Ba2+ on [Ca2+]i and [3H]NE release that Ba2+ stimulates exocytosis by a Ca(2+)-dependent mechanism. The Ba(2+)-induced rise in [Ca2+]i is a result of two separate actions: (i) the release of Ca2+ f...
Ethanol, added to primary cultures of cerebellar granule neurons simultaneously with NMDA, was pr... more Ethanol, added to primary cultures of cerebellar granule neurons simultaneously with NMDA, was previously shown to inhibit the anti-apoptotic effect of NMDA. The in vitro anti-apoptotic effect of NMDA is believed to mimic in vivo protection against apoptosis afforded by innervation of developing cerebellar granule neurons by glutamatergic mossy fibers. Therefore, the results suggested that the presence of ethanol in the brain at a critical period of development would promote apoptosis. In the present studies, we examined the effect of chronic ethanol exposure on the anti-apoptotic action of NMDA in cerebellar granule neurons. The neurons were treated with ethanol in vitro for 1-3 days in the absence of NMDA. Even after ethanol was removed from the culture medium, as ascertained by gas chromatography, the protective effect of added NMDA was significantly attenuated. The decreased anti-apoptotic effect of NMDA was associated with a change in the properties of the NMDA receptor, as indicated by a decrease in ligand binding, decreased expression of NMDA receptor subunit proteins, and decreased functional responses including stimulation of increases in intracellular Ca(2+) and induction of brain-derived neurotrophic factor expression. The latter effect may directly underlie the attenuated protective effect of NMDA in these neurons. The results suggest that ethanol exposure during development can have long-lasting effects on neuronal survival. The change in the NMDA receptor caused by chronic ethanol treatment may contribute to the loss of cerebellar granule neurons that is observed in animals and humans exposed to ethanol during gestation.
The effects of various neurotransmitters on phosphoinositide hydrolysis, mobilization of Ca2+ and... more The effects of various neurotransmitters on phosphoinositide hydrolysis, mobilization of Ca2+ and release of [3H]-norepinephrine ([3H]-NE) were studied in cultures of sympathetic neurons of chick embryos. [3H]-inositol-1,4,5-triphosphate ([3H]-IP3) was increased in sympathetic neurons by acetylcholine (ACh), muscarine and serotonin (5-HT). Dopamine and norepinephrine did not stimulate phosphoinositide hydrolysis. Intracellular concentration of free Ca2+ ([Ca2+]i) was measured in Indo-1-loaded sympathetic neurons at rest and after addition of test agents. Measurements were made in the cell body and growth cone regions since Ca2+ mobilization is known to be different in different regions of the sympathetic neurons. ACh (nicotinic component was blocked by hexamethonium) and 5-HT failed to increase the [Ca2+]i, in the cell body as well as in the growth cone. The spontaneous release of [3H]-NE was not affected by ACh and 5-HT. Caffeine increased the [Ca2+]i only in the cell body but not ...
The Journal of pharmacology and experimental therapeutics, 2000
A novel series of N-substituted 4-ureido-5,7-dichloro-quinolines were synthesized to contain phar... more A novel series of N-substituted 4-ureido-5,7-dichloro-quinolines were synthesized to contain pharmacophores directed at voltage-sensitive sodium channels (VSNaCs) and N-methyl-D-aspartate (NMDA) receptors. These compounds were shown to act in a use-dependent manner as antagonists of VSNaCs and to act as selective competitive antagonists at the strychnine-insensitive glycine recognition site of NMDA receptors. These agents had little or no effect on alpha-adrenergic receptors, other glutamate receptors, or sites other than the glycine site on the NMDA receptor, and did not block voltage-sensitive calcium channels in vitro. In vivo, the compounds were active in preventing or reducing the signs and symptoms of neurohyperexcitability and had anxiolytic properties. Unlike benzodiazepines, N-substituted 4-ureido-5, 7-dichloro-quinolines showed little interaction with the sedative effects of ethanol, but were effective in controlling ethanol withdrawal seizures. The combined actions of the...
Alcohol research & health : the journal of the National Institute on Alcohol Abuse and Alcoholism, 2008
Researchers from a wide variety of backgrounds and with a broad range of goals have utilized high... more Researchers from a wide variety of backgrounds and with a broad range of goals have utilized high-throughput screening technologies (i.e., microarray technologies) to identify candidate genes that may be associated with an observable characteristic or behavior (i.e., phenotype) of interest. However, the initial microarray analyses typically also yield many genes that are not related to the phenotype of interest. Therefore, additional analyses are necessary to select the most likely candidates and eventually identify one or more genes that actually underlie that phenotype. After briefly explaining how microarray data are generated, this article describes one approach to narrowing down the resulting candidate genes and a database that can help in this analysis.
Alcohol research & health : the journal of the National Institute on Alcohol Abuse and Alcoholism, 2008
The development of alcohol dependence is posited to involve numerous changes in brain chemistry (... more The development of alcohol dependence is posited to involve numerous changes in brain chemistry (i.e., neurotransmission) that lead to physiological signs of withdrawal upon abstinence from alcohol as well as promote vulnerability to relapse in dependent people. These neuroadaptive changes often occur in those brain neurotransmission systems that are most sensitive to the acute, initial effects of alcohol and/or contribute to a person’s initial alcohol consumption. Studies of these neuroadaptive changes have been aided by the development of animal models of alcohol dependence, withdrawal, and relapse behavior. These animal models, as well as findings obtained in humans, have shed light on the effects that acute and chronic alcohol exposure have on signaling systems involving the neurotransmitters glutamate, γaminobutyric acid (GABA), dopamine, and serotonin, as well as on other signaling molecules, including endogenous opioids and corticotrophin-releasing factor (CRF). Adaptation to...
Alcohol and alcoholism (Oxford, Oxfordshire). Supplement, 1994
Ethanol inhibits the function of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor in... more Ethanol inhibits the function of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor in various neuronal systems, but the mechanism of the inhibition has not been elucidated. Previous work, using primary cultures of rat cerebellar granule cells, showed that both exposure to alcohol and activation of protein kinase C (PKC) by the phorbol ester PMA reduced the potency of the co-agonist, glycine, to enhance NMDA receptor function (measured as an increase in intracellular Ca2+), resulting in inhibition of the NMDA response at low glycine concentrations. Inhibition of NMDA receptor function by PMA and ethanol could also be overcome by PKC antagonists, implicating PKC in the inhibitory effect of ethanol. We have now compared the effects of ethanol and PKC activation of NMDA receptor function in primary cultures of rat cerebral cortical cells. The receptor in these cells was much less sensitive to ethanol inhibition, and the inhibition was not overcome by high concentrations of g...
Proceedings of the National Academy of Sciences, 1993
The concentration of cytosolic free Ca2+ ([Ca2+]i) and the release of tritiated norepinephrine ([... more The concentration of cytosolic free Ca2+ ([Ca2+]i) and the release of tritiated norepinephrine ([3H]NE) were monitored during Ba2+ stimulation of sympathetic neurons cultured from chick embryos. Ba2+ (2.5 mM in Ca(2+)-free medium) caused a rise in [Ca2+]i in all regions (cell bodies, neurites, and growth cones) of sympathetic neurons and evoked [3H]NE release in the absence of other stimuli. The increase in [Ca2+]i and release of [3H]NE were sustained for up to 30 min in the presence of Ba2+. When Ba(2+)-stimulated cells were immediately washed in Ca(2+)-free Ba(2+)-free EGTA solution, both the elevated [Ca2+]i and [3H]NE release returned to basal levels, with similar, fast, time courses. Ba2+ also blocked Ca2+ efflux from neurons loaded with 45Ca. We conclude from the parallel effects of Ba2+ on [Ca2+]i and [3H]NE release that Ba2+ stimulates exocytosis by a Ca(2+)-dependent mechanism. The Ba(2+)-induced rise in [Ca2+]i is a result of two separate actions: (i) the release of Ca2+ f...
Ethanol, added to primary cultures of cerebellar granule neurons simultaneously with NMDA, was pr... more Ethanol, added to primary cultures of cerebellar granule neurons simultaneously with NMDA, was previously shown to inhibit the anti-apoptotic effect of NMDA. The in vitro anti-apoptotic effect of NMDA is believed to mimic in vivo protection against apoptosis afforded by innervation of developing cerebellar granule neurons by glutamatergic mossy fibers. Therefore, the results suggested that the presence of ethanol in the brain at a critical period of development would promote apoptosis. In the present studies, we examined the effect of chronic ethanol exposure on the anti-apoptotic action of NMDA in cerebellar granule neurons. The neurons were treated with ethanol in vitro for 1-3 days in the absence of NMDA. Even after ethanol was removed from the culture medium, as ascertained by gas chromatography, the protective effect of added NMDA was significantly attenuated. The decreased anti-apoptotic effect of NMDA was associated with a change in the properties of the NMDA receptor, as indicated by a decrease in ligand binding, decreased expression of NMDA receptor subunit proteins, and decreased functional responses including stimulation of increases in intracellular Ca(2+) and induction of brain-derived neurotrophic factor expression. The latter effect may directly underlie the attenuated protective effect of NMDA in these neurons. The results suggest that ethanol exposure during development can have long-lasting effects on neuronal survival. The change in the NMDA receptor caused by chronic ethanol treatment may contribute to the loss of cerebellar granule neurons that is observed in animals and humans exposed to ethanol during gestation.
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